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Primary cilia are solitary, immotile sensory organelles present on most cells in the body that participate broadly in human health, physiology and disease. Cilia generate a unique environment for signal transduction with tight control of protein, lipid and second messenger concentrations within a relatively small compartment, enabling reception, transmission and integration of biological information. In this Review, we discuss how cilia function as signalling hubs in cell–cell communication using three signalling pathways as examples: ciliary G-protein-coupled receptors (GPCRs), the Hedgehog (Hh) pathway and polycystin ion channels. We review how defects in these ciliary signalling pathways lead to a heterogeneous group of conditions known as ‘ciliopathies’, including metabolic syndromes, birth defects and polycystic kidney disease. Emerging understanding of these pathways’ transduction mechanisms reveals common themes between these cilia-based signalling pathways that may apply to other pathways as well. These mechanistic insights reveal how cilia orchestrate normal and pathophysiological signalling outputs broadly throughout human biology.Cilia are microtubule-based cell projections that provide a unique environment with precise protein, lipid and second messenger concentrations, thereby creating specialized signalling hubs. This Review discusses recent multidisciplinary, mechanistic insights into cilia-based signalling pathways during development and homeostasis.

The primary cilium is an antenna-like, immotile organelle present on most types of mammalian cells, which interprets extracellular signals that regulate growth and development. Although once considered a vestigial organelle, the primary cilium is now the focus of considerable interest. We now know that ciliary defects lead to a panoply of human diseases, termed ciliopathies, and the loss of this organelle may be an early signature event during oncogenic transformation. Ciliopathies include numerous seemingly unrelated developmental syndromes, with involvement of the retina, kidney, liver, pancreas, skeletal system and brain. Recent studies have begun to clarify the key mechanisms that link cilium assembly and disassembly to the cell cycle, and suggest new possibilities for therapeutic intervention. Sanchez and Dynlacht discuss recent insights into the mechanisms of primary cilia assembly and disassembly, and the relationships between ciliogenesis and cell cycle regulation as well as disease.

Key Points Multiciliated cells are derived from progenitor cells after the inhibition of Notch signalling and activation of two coiled-coil domain-containing proteins: geminin coiled-coil domain-containing protein 1 (GEMC1) and multicilin. During multiciliated cell differentiation, tens of centrioles are produced by harnessing the signalling pathway that is used for canonical centriole duplication in dividing cells. A combination of cytoskeletal networks, planar cell polarity pathways and mechanical forces are involved in the coordinated beating of multiple motile cilia. Multiciliated cells fulfil diverse and crucial functions such as protection from external particles, active transport of materials and regulation of organ homeostasis. Multiciliated cells line the lumen of the vertebrate central nervous system and respiratory and reproductive tracts, where the unidirectional beating of cilia assemblies supports the polarized flow of fluids or movement of cells or particles. Recent studies shed new light on how multiciliated cells arise and how they function. Multiciliated cells are epithelial cells that are in contact with bodily fluids and are required for the proper function of major organs including the brain, the respiratory system and the reproductive tracts. Their multiple motile cilia beat unidirectionally to remove particles of external origin from their surface and/or drive cells or fluids into the lumen of the organs. Multiciliated cells in the brain are produced once, almost exclusively during embryonic development, whereas in respiratory tracts and oviducts they regenerate throughout life. In this Review, we provide a cell-to-organ overview of multiciliated cells and highlight recent studies that have greatly increased our understanding of the mechanisms driving the development and function of these cells in vertebrates. We discuss cell fate determination and differentiation of multiciliated cells, and provide a comprehensive account of their locations and functions in mammals.

Key Points Cilia are complex sensory and motile organelles found on almost all cells of the body. The complexity of the cilium raises the question of how it is built in an orderly fashion. Ciliary assembly proceeds in a stepwise manner: centrioles form basal bodies, dock on the cortex and induce outgrowth of the cilium. Assembly also involves protein-trafficking from the cytoplasm to the base of the cilium and selective import of ciliary proteins through a channel that may be analogous to the nuclear pore complex. Sustained growth of cilia requires active transport, which is provided by the intraflagellar transport (IFT) system. Assembly of cilia is a function of cell cycle stage and is tightly regulated to control the length of the final structure. Ciliary length seems to result from a continuous steady-state balance of assembly and disassembly, with the inherent length-dependence of IFT-mediated transport leading to a length-dependent assembly rate. Cilium assembly requires the coordination of motor-driven intraflagellar transport, membrane trafficking and import of cilium-specific proteins through a barrier at the ciliary transition zone. Recent findings provide insights into how cilia might assemble and disassemble in synchrony with the cell cycle and achieve a steady-state length. The cilium is a complex organelle, the assembly of which requires the coordination of motor-driven intraflagellar transport (IFT), membrane trafficking and selective import of cilium-specific proteins through a barrier at the ciliary transition zone. Recent findings provide insights into how cilia assemble and disassemble in synchrony with the cell cycle and how the balance of ciliary assembly and disassembly determines the steady-state ciliary length, with the inherent length-dependence of IFT rendering the ciliary assembly rate a decreasing function of length. As cilia are important in sensing and processing developmental signals and directing the flow of fluids such as mucus, defects in ciliogenesis and length control are likely to underlie a range of cilium-related human diseases.

Distal appendages (DAPs) are nanoscale, pinwheel-like structures protruding from the distal end of the centriole that mediate membrane docking during ciliogenesis, marking the cilia base around the ciliary gate. Here we determine a super-resolved multiplex of 16 centriole-distal-end components. Surprisingly, rather than pinwheels, intact DAPs exhibit a cone-shaped architecture with components filling the space between each pinwheel blade, a new structural element we term the distal appendage matrix (DAM). Specifically, CEP83, CEP89, SCLT1, and CEP164 form the backbone of pinwheel blades, with CEP83 confined at the root and CEP164 extending to the tip near the membrane-docking site. By contrast, FBF1 marks the distal end of the DAM near the ciliary membrane. Strikingly, unlike CEP164, which is essential for ciliogenesis, FBF1 is required for ciliary gating of transmembrane proteins, revealing DAPs as an essential component of the ciliary gate. Our findings redefine both the structure and function of DAPs. Distal appendages (DAPs) at the cilia base mediate membrane docking during ciliogenesis. Here the authors use super-resolution microscopy to map 16 centriole distal end components, revealing the structure of the backbone of the DAP, as well as a previously undescribed distal appendage matrix.

The canonical mitotic cell cycle coordinates DNA replication, centriole duplication and cytokinesis to generate two cells from one 1 . Some cells, such as mammalian trophoblast giant cells, use cell cycle variants like the endocycle to bypass mitosis 2 . Differentiating multiciliated cells, found in the mammalian airway, brain ventricles and reproductive tract, are post-mitotic but generate hundreds of centrioles, each of which matures into a basal body and nucleates a motile cilium 3 , 4 . Several cell cycle regulators have previously been implicated in specific steps of multiciliated cell differentiation 5 , 6 . Here we show that differentiating multiciliated cells integrate cell cycle regulators into a new alternative cell cycle, which we refer to as the multiciliation cycle. The multiciliation cycle redeploys many canonical cell cycle regulators, including cyclin-dependent kinases (CDKs) and their cognate cyclins. For example, cyclin D1, CDK4 and CDK6, which are regulators of mitotic G1-to-S progression, are required to initiate multiciliated cell differentiation. The multiciliation cycle amplifies some aspects of the canonical cell cycle, such as centriole synthesis, and blocks others, such as DNA replication. E2F7, a transcriptional regulator of canonical S-to-G2 progression, is expressed at high levels during the multiciliation cycle. In the multiciliation cycle, E2F7 directly dampens the expression of genes encoding DNA replication machinery and terminates the S phase-like gene expression program. Loss of E2F7 causes aberrant acquisition of DNA synthesis in multiciliated cells and dysregulation of multiciliation cycle progression, which disrupts centriole maturation and ciliogenesis. We conclude that multiciliated cells use an alternative cell cycle that orchestrates differentiation instead of controlling proliferation. A distinct cell cycle redeploys many canonical cell cycle regulators to control the differentiation of multiciliated cells, with the transcription factor E2F7 playing a pivotal part in this modified cell cycle.

Mammalian Hedgehog (Hh) signal transduction requires a primary cilium, a microtubule-based organelle, and the Gli–Sufu complexes that mediate Hh signalling, which are enriched at cilia tips. Kif7, a kinesin-4 family protein, is a conserved regulator of the Hh signalling pathway and a human ciliopathy protein. Here we show that Kif7 localizes to the cilium tip, the site of microtubule plus ends, where it limits cilium length and controls cilium structure. Purified recombinant Kif7 binds the plus ends of growing microtubules in vitro , where it reduces the rate of microtubule growth and increases the frequency of microtubule catastrophe. Kif7 is not required for normal intraflagellar transport or for trafficking of Hh pathway proteins into cilia. Instead, a central function of Kif7 in the mammalian Hh pathway is to control cilium architecture and to create a single cilium tip compartment, where Gli–Sufu activity can be correctly regulated. Anderson and colleagues report that the kinesin-4 family member Kif7 binds to microtubule plus ends at cilium tips to regulate their length and structure, and to ensure the fidelity of Hedgehog signalling.

Distal appendages (DAPs) are vital in cilia formation, mediating vesicular and ciliary docking to the plasma membrane during early ciliogenesis. Although numerous DAP proteins arranging a nine-fold symmetry have been studied using superresolution microscopy analyses, the extensive ultrastructural understanding of the DAP structure developing from the centriole wall remains elusive owing to insufficient resolution. Here, we proposed a pragmatic imaging strategy for two-color single-molecule localization microscopy of expanded mammalian DAP. Importantly, our imaging workflow enables us to push the resolution limit of a light microscope well close to a molecular level, thus achieving an unprecedented mapping resolution inside intact cells. Upon this workflow, we unravel the ultra-resolved higher-order protein complexes of the DAP and its associated proteins. Intriguingly, our images show that C2CD3, microtubule triplet, MNR, CEP90, OFD1, and ODF2 jointly constitute a unique molecular configuration at the DAP base. Moreover, our finding suggests that ODF2 plays an auxiliary role in coordinating and maintaining DAP nine-fold symmetry. Together, we develop an organelle-based drift correction protocol and a two-color solution with minimum crosstalk, allowing a robust localization microscopy imaging of expanded DAP structures deep into the gel-specimen composites. The authors have combined direct stochastic optical reconstruction microscopy with expansion microscopy to describe the 3-dimensional molecular organization of centriolar distal appendages.

The ability to sense and respond to mechanical stimuli emanates from sensory neurons and is shared by most, if not all, animals. Exactly how such neurons receive and distribute mechanical signals during touch sensation remains mysterious. Here, we show that sensation of mechanical forces depends on a continuous, pre-stressed spectrin cytoskeleton inside neurons. Mutations in the tetramerization domain of Caenorhabditis elegans β-spectrin (UNC-70), an actin-membrane crosslinker, cause defects in sensory neuron morphology under compressive stress in moving animals. Through atomic force spectroscopy experiments on isolated neurons, in vivo laser axotomy and fluorescence resonance energy transfer imaging to measure force across single cells and molecules, we show that spectrin is held under constitutive tension in living animals, which contributes to elevated pre-stress in touch receptor neurons. Genetic manipulations that decrease such spectrin-dependent tension also selectively impair touch sensation, suggesting that such pre-tension is essential for efficient responses to external mechanical stimuli. How sensory neurons integrate mechanical signals during touch sensation has remained unclear. Using a combination of laser axotomy and FRET imaging to measure force across single cells and molecules, Goodman and colleagues show that the neuronal spectrin cytoskeleton transduces touch sensation in C. elegans .

Epithelial tissues provide front-line barriers shielding the organism from invading pathogens and harmful substances. In the airway epithelium, the combined action of multiciliated and secretory cells sustains the mucociliary escalator required for clearance of microbes and particles from the airways. Defects in components of mucociliary clearance or barrier integrity are associated with recurring infections and chronic inflammation. The timely and balanced differentiation of basal cells into mature epithelial cell subsets is therefore tightly controlled. While different growth factors regulating progenitor cell proliferation have been described, little is known about the role of metabolism in these regenerative processes. Here we show that basal cell differentiation correlates with a shift in cellular metabolism from glycolysis to fatty acid oxidation (FAO). We demonstrate both in vitro and in vivo that pharmacological and genetic impairment of FAO blocks the development of fully differentiated airway epithelial cells, compromising the repair of airway epithelia. Mechanistically, FAO links to the hexosamine biosynthesis pathway to support protein glycosylation in airway epithelial cells. Our findings unveil the metabolic network underpinning the differentiation of airway epithelia and identify novel targets for intervention to promote lung repair. Airway epithelial repair, a key process in the recovery from lung injury, requires a metabolic shift from glycolysis to fatty acid oxidation (FAO). Pharmacological FAO promotion enhances epithelial differentiation, suggesting new therapeutic options.